The overall goal of the proposed studies in this R21 application is a better understanding of the molecular and cellular mechanisms by which aldosterone regulates sodium homeostasis. Studies by others and us revealed that serum- and glucocorticoid-induced kinase-1 (SGK1), an early aldosterone-induced gene, plays an important role in mediating the effects of aldosterone on Na transport. The mechanism through which SGK1 alters Na homeostasis in vivo is still not entirely clear. Our recent studies indicate that NDRG1 (N-myc down-regulated gene-1) is the dominant SGK1 substrate in the kidney and in cultured cortical collecting duct (CCD) cells, and that activation of the mineralocorticoid receptor (MR) but not of the glucocorticoid receptor (GR) results in an SGK1-dependent hyperphosphorylation on NDRG1. NDRG1 is highly expressed in the aldosterone-sensitive nephron segments. Based on these observations, we hypothesize that NDRG1 and its MR-specific regulation are involved in aldosterone-induced Na transport. In Aim 1, we will determine the contribution of NDRG1 to aldosterone-stimulated Na transport in vivo, by using NDRG1 knockout mice and comparing renal Na handling of such mice to that of wild type mice. These studies will be complemented by experiments aimed at exploring the cellular mechanism of action of NDRG1 and its relationship with SGK1 in the regulation of Na transport, using CCD cell lines with inducible deletion of NDRG1. Aim 2 is to identify the MR-specific regulatory event that leads to enhanced SGK1-mediated phosphorylation of NDRG1 in CCD cells. Our hypothesis is that engagement of MR (but not GR) results in increased activity of the mammalian target of rapamycin (mTOR) Complex 2 (mTORC2), which is a key regulator of SGK1 activity. We will test this hypothesis by (a) determining the effect of MR- vs. GRactivation on the activity of mTORC2 in intact CCD cells;(b) examining the underlying mechanism by determining the phosphorylation status of mTORC2 and searching for the upstream kinase(s) that activate(s) mTORC2 following MR vs. GR engagement. If our hypotheses are correct, the proposed studies should not only lead to a better understanding of the molecular mechanism by which aldosterone and SGK1 regulates Na homeostasis but should also have a more direct clinical relevance. MR-selective mTOR activation would be an exciting discovery since Mtor plays a central role in the regulation of many pathways involved in MR-mediated diseases in non-traditional target cells, such as cardiac fibrosis, cardiac hypertrophy and atherosclerosis PUBLIC HEALTH RELEVANCE: This new application is aimed at a better understanding of the molecular and cellular mechanisms by which aldosterone, a hormone produced by the adrenal glands, regulates sodium homeostasis in the body. We propose to perform both experiments in mice as well as in isolated cultured kidney cells to study the role of one particular protein, called NDRG1 in ion transport by the kidney. Furthermore, we are trying to understand the mechanisms by which different steroid hormone receptors exert specific actions on the kidney.